脂肪间充质干细胞对脊髓脊膜膨出胎鼠脊髓和膀胱损伤的修复作用

Therapeutic effects of adipose-derived mesenchymal stem cells on spinal cord injury and bladder dysplasia in fetal rats with meningomyelocele

目的探索脂肪间充质干细胞(adipose-derived mesenchymal stem cells,ADSCs)在脊髓脊膜膨出中的治疗潜能。方法贴壁法提取和培养SD雄鼠ADSCs,流式细胞术鉴定表面标志物表达,并鉴定其向成脂和成骨细胞分化的潜能。8只孕鼠在孕10 d接受全反式维甲酸灌胃处理,孕15 d时向每个羊膜腔内交替注射ADSCs细胞悬液或PBS。孕21 d将有脊髓脊膜膨出(meningomyelocele,MMC)缺损的胎鼠根据羊膜腔内注射液体分为实验组(注射ADSCs,36只)和对照组(注射PBS,24只),记录并比较胎鼠顶臀长和体重数据及骶尾部皮肤缺损面积。用蛋白质印迹法检测不同组胎鼠脊髓组织中NeuN、PCNA、Cleaved caspase 3、IL-1β、GFAP和Iba-1蛋白表达水平以及膀胱组织中αSMA、SMMHC、Cx43和β3-微管蛋白表达水平。实验组和对照组结果比较采用t检验,P<0.05为差异具有统计学意义。结果ADSCs镜下呈纺锤形,贴壁生长,表面阳性表达CD29、CD44、CD73和CD90,阴性表达CD11b/c、CD34和CD45,符合间充质干细胞表面标志物表达特点;可成功诱导为成脂和成骨细胞。羊膜腔内注射ADSCs后,MMC胎鼠骶尾部皮肤缺损面积显著减小,相较于对照组,实验组胎鼠脊髓组织中GFAP、Iba-1和IL-1β蛋白表达水平显著下调;膀胱组织中Cx43蛋白表达水平显著上调,两组胎鼠脊髓和膀胱组织中其余蛋白表达水平无差异。结论羊膜腔内注射ADSCs治疗可为干细胞胎内治疗MMC及继发的膀胱功能障碍提供有益探索。

Objective To explore the therapeutic potential of adipose-derived mesenchymal stem cells(ADSCs)in meningomyelocele.Methods ADSCs from Sprague-Dawley(SD)male rats were extracted and cultured by adherent method.Surface marker expression was identified by flow cytometry and their potential of differentiating into adipoblasts and osteoblasts was identified.Eight pregnant rats received all-trans retinoic acid by gavage at gestational day 10 and ADSCs or phosphate-buffered saline(PBS)were alternately injected into each amniotic cavity at gestational day 15.Fetuses with meningomyelocele(MMC)defects were divided into two groups of experimental(injected with ADSCs,n=36)and control(injected with PBS,n=24)according to intra-amniotic fluid injection at gestational day 21.Crown-rump length,body weight and sacrococcygeal skin defect area were compared.Protein expression levels of Neuronal nuclei,proliferating cell nuclear antigen(PCNA),cleaved caspase 3,interleukin-1β(IL-1β),glial fibrillary acidic protein(GFAP)and ionized calcium binding adaptor molecule 1(Iba-1)in spinal cord tissues in different groups as well as protein expression levels ofαSMA,SMMHC,Cx43 andβ3 tubulin in bladder tissues were detected by Western blot.The results of two groups were compared by t-test and P<0.05 was deemed as statistically significant.Results ADSCs were spindle-shaped,adherent and growing with a positive expression of CD29,CD44,CD73 and CD90 and a negative expression of CD11b/c,CD34 and CD45 on surface.The pattern was consistent with the expression characteristics of surface markers of mesenchymal stem cells.And they could be successfully induced into adipoblasts and osteoblasts.After intra-amniotic injection of ADSCs,sacrococcygeal skin defect area decreased markedly.As compared with control group,protein expression levels of GFAP,Iba-1 and IL-1βin spinal cord tissue were markedly down-regulated in experimental group;protein expression level of Cx43 in bladder tissue was significantly up-regulated and no inter-group difference existed in the expression levels of other proteins in spinal cord and bladder tissue.Conclusions Intra-amniotic injection of ADSCs therapy can provide an effective intrauterine treatment of MMC and secondary bladder dysfunction with stem cells.